Type 1 fimbria and P pili: regulatory mechanisms of the prototypical members of the chaperone-usher fimbrial family

Adherence to both cellular and abiotic surfaces is a crucial step in the interaction of bacterial pathogens and commensals with their hosts. Bacterial surface structures known as fimbriae or pili play a fundamental role in the early colonization stages by providing specificity or tropism. Among the various fimbrial families, the chaperone-usher family has been extensively studied due to its ubiquity, diversity, and abundance. This family is named after the components that facilitate their biogenesis. Type 1 fimbria and P pilus, two chaperone-usher fimbriae associated with urinary tract infections, have been thoroughly investigated and serve as prototypes that have laid the foundations for understanding the biogenesis of this fimbrial family. Additionally, the study of the mechanisms regulating their expression has also been a subject of great interest, revealing that the regulation of the expression of the genes encoding these structures is a complex and diverse process, involving both common global regulators and those specific to each operon.


Introduction
Fimbriae, also known as pili, are filamentous protein appendages that extend beyond the cell surface and have a diameter of 2-8 nm and variable length.These structures may consist of hundreds of copies of a single protein, the major fimbrial subunit, also called pilin.Alternatively, fimbriae may be composed of a pilin filament tipped by additional proteins known as minor fimbrial subunits or adhesins, which mediate interactions with surface receptors (Hospenthal et al. 2017).Fimbriae also play roles in attachment to abiotic and under different conditions have been described (Clegg et al. 2011;Gahlot et al. 2022).This review focuses on providing an overview of the established regulatory pathways involved in the transcriptional control of the prototypical C-U fimbrial operons, fim and pap, which have served as pivotal models in decades of research.

Type 1 fimbria of E. coli
The Type 1 Fimbria (T1F), also abbreviated as Fim, represents one of the most well-studied members of the C-U assembly family.Widely distributed in E. coli, T1F plays a critical role in urinary tract infections (UTIs).T1F structure, biogenesis and regulation have been extensively investigated (Behzadi 2020;Bessaiah et al. 2021;Hospenthal and Waksman 2019;Lillington et al. 2015;Wurpel et al. 2013).
UTIs are among the most common community and hospital-acquired infectious diseases, ranging from uncomplicated cystitis to potentially severe conditions such as pyelonephritis and septicemia, depending on host-associated risk factors (Klein and Hultgren 2020).A predominant cause of UTIs is uropathogenic E. coli (UPEC), which can ascend to the kidneys upon colonizing the urethra and the bladder.After initial contact with the bladder lumen, the bacteria use the T1F to adhere to mannosylated glycoproteins on the surface of the urothelial epithelium, facilitating colonization (Pullanhi et al. 2019;Wu et al. 1996).Furthermore, T1F also enables UPEC to establish intracellular bacterial communities (IBCs) and form biofilms, contributing to the persistence and pathogenesis of UTIs (Reisner et al. 2014;Rosen et al. 2007).
T1F is a hair-like structure, typically 1-2 μm in length, characterized by a thin tip fibrillum mounted on a longer and thicker helical rod.The genes responsible for T1F synthesis are encoded in the chromosomal fim gene cluster.The fim operon comprises seven genes involved in pilus assembly.Beginning with fimA, which encodes the major fimbrial subunit or pilin, the operon sequentially includes fimI, encoding a putative anchor subunit, fimC a periplasmic chaperone, fimD the usher, fimF and fimG tip adaptor subunits and, finally, fimH encoding the tip adhesin (Hospenthal and Waksman 2019).
The regulation of T1F expression involves a sophisticated regulatory network centered around fimS, a 314-base pair invertible DNA segment containing the promoter of the fim operon.Upstream of fimS are located the genes encoding FimB and FimE (Klemm 1986), two tyrosine-dependent site-specific recombinases that bind to nonidentical recombinase-binding elements flanking two 9 bp inverted repeat sequences (IRR and IRL), which control the orientation of fimS.Transcription of the fim operon occurs when fimS is in the phase-ON orientation, but not when this promoter region is inverted to the phase-OFF orientation.FimB can invert the switch with similar frequency in both directions (ONto-OFF and OFF-to-ON), whereas FimE predominantly inverts it towards the ON-to-OFF direction (McCusker et al. 2008) (Fig. 1).
The optimal expression of the FimB and FimE recombinases is essential for regulating T1F phase variation in response to growth conditions.Several factors influence the expression of fimB, for which three promoters have been reported (Schwan et al. 1994).Negatively, the stationary phase σ factor (RpoS), as cells enter the stationary phase (Dove et al. 1997).The response regulator OmpR primarily represses fimB under low pH-high osmolarity conditions (Rentschler et al. 2013;Schwan et al. 2002), while MarA represses it in the presence of salicylate (Vila and Soto 2012).Conversely, the sialic acid transcriptional regulator (NanR) and the N-acetylglucosamine repressor (NagC) prevent fimB repression by DAM methylation (Sohanpal et al. 2004).
Studies have shown that the loss of the cpxRA genes, which encode the two-component system (TCS) activated under membrane stress, reduces the phase-ON state, suggesting that CpxR positively regulates the fimB promoter (Miki et al. 2024).RcsB, the response regulator of the Rcs phosphorelay system (Schwan et al. 2007), the MarA-like transcriptional regulator SlyA (McVicker et al. 2011) and the regulatory alarmone guanosine tetraphosphate (ppGpp), along with its DksA cofactor (Aberg et al. 2008), all play positive roles in fimB regulation.In addition, the LysR-type regulator LrhA (Blumer et al. 2005) and, under iron-limiting conditions, the iron-sulfur cluster regulator IscR (Wu and Outten 2009), activate fimE transcription, while RcsB represses it (Schwan et al. 2007).
Furthermore, recombination directionality factors are also involved in fimS switching.The integration host factor (IHF) and the leucine-responsive regulatory protein (Lrp) promote the phase-ON orientation by facilitating a recombination-proficient structure.IHF binds with high affinity to two sites, one adjacent to fimS (site I) and another within the invertible element (site II), while Lrp binds to three sites within the same element (Blomfield et al. 1997;Roesch and Blomfield 1998).In contrast, the histone-like nucleoidstructuring protein (H-NS) maintains fimS in the phase-OFF position by directly binding to the fimB promoter region and segments adjacent to and within fimS (Corcoran and Dorman 2009;Donato et al. 1997).Additionally, the factor for inversion stimulation (Fis), another nucleoid-associated protein, plays a complex and seemingly opposite regulatory role in T1F expression.In the presence of the recombinase FimE, Fis promotes the OFF orientation of fimS (Saldana-Ahuactzi et al. 2022).Conversely, under conditions that relax DNA 1 3 supercoiling, such as late exponential and stationary phases, Fis biases recombinase FimB towards the ON phase by binding to the LRP-2 site (Conway et al. 2023).However, under iron-rich conditions, Fur binds to the same LRP-2 site and represses fim expression (Kurabayashi et al. 2016).
Other site-specific recombinases that participate in controlling the fimS switch have been described in E. coli.HbiF (Xie et al. 2006) and IpbA (Bryan et al. 2006) predominantly switch from OFF to ON, while IpuA (Bryan et al. 2006) and FimX (Hannan et al. 2008) have FimB-like ON-to-OFF and OFF-to-ON activity.
Additional factors positively influence the activity of the fimA promoter, including YqhG, a predicted periplasmic protein (Bessaiah et al. 2019), the QseBC two-component system (TCS) (Gou et al. 2019) and FNR, a well-known global regulator (Barbieri et al. 2014).On the other hand, the second messenger cyclic AMP (adenosine monophosphate) and the cAMP receptor protein (CRP) play a dual role in type 1 fimbriation, negatively affecting both the phase variation and fimA promoter activity (Muller et al. 2009).Additionally, the inactivation of the phosphate-specific transport (Pst) system constitutively activates the TCS PhoBR, which mediates the activation of YaiC, a diguanylate cyclase (DGC), which in turn increases the accumulation of bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) and represses the fim operon (Crepin et al. 2017).LeuO, a LysR-type transcriptional regulator, acts as a negative regulator (Shimada et al. 2011).During the formation of intracellular bacterial communities, which are associated with UTIs, UPEC overexpresses yeaR, which encodes a protein associated with resistance to oxidative stress that seems to indirectly reduce T1F expression (Conover et al. 2016).
Furthermore, various sRNAs post-transcriptionally regulate fim expression.For instance, RseX, an sRNA associated with envelope stress, interacts with the fimB mRNA, resulting in downregulation of fim expression (Mihailovic et al. 2021).A comprehensive screening of a plasmid collection that overexpressed different small RNAs revealed several negative regulators of T1F synthesis, including DsrA, FimE DNA recombinases.For Salmonella, the production of T1F is also regulated by a phase-variable mechanism characterized by in vitro switching between a poorly fimbriated state on solid media and a highly fimbriated state when the bacteria grow in static liquid media (Old and Duguid 1970).However, unlike in E. coli, the expression of this fim operon is not controlled by DNA inversion; the promoter region is oriented in direction to promote fimA transcription, and there are no apparent homologs of FimB and FimE encoded within the gene cluster (Clegg et al. 1996).The Salmonella fimA promoter is inactive in E. coli, indicating that this bacterium does not have all the elements needed to activate the expression of T1F in Salmonella (Yeh et al. 1995).Similarly, no genes encoding regulatory proteins related to FimZ, FimY, or FimW were found within the E. coli fim gene cluster, indicating that, despite relatedness, the regulatory mechanisms are different.
FimZ, FimY, and FimW are the three major regulatory proteins for T1F in Salmonella and are encoded by individual genes that are expressed under their own promoters (Tinker et al. 2001); they control the fim operon expression primarily through regulation of the fimA promoter (Tinker and Clegg 2000;Tinker et al. 2001;Yeh et al. 1995) (Fig. 2).
FimZ is an orphan 25-kDa response regulator associated with TCSs, which phosphorylation is necessary for the activation of fimA (Zeiner et al. 2013).FimZ is the dominant transcriptional activator for expression of T1F, even under conditions favoring fimbriation (Yeh et al. 1995), and it binds to the region upstream (from − 47 to -98 nucleotides) of the fimA transcription initiation site (Yeh et al. 2002).The communication between fimbriae and flagellar expression in enterobacteria seems to be a common phenomenon in motile organisms.In S. Typhimurium, FimZ is one of the connections, as its overexpression is also associated with a decrease in motility even in non-fimbriate mutants.It is hypothesized that FimZ could be part of a signaling pathway facilitating coordination between adherence and dissemination (Clegg and Hughes 2002).
FimZ works in cooperation with FimY, a LuxR-like domain-containing protein encoded in the vicinity of the fimZ gene in S. Typhimurium (Tinker and Clegg 2000).This regulator also possesses DNA-binding capacity and binds to a lux box sequence within the fimZ promoter, but no interaction has been found for the fimA, fimY, or fimW promoters (Wang et al. 2014).Overproduction of FimY cannot restore the fimbriae synthesis in a FimZ mutant, but high levels of FimZ can overcome the non-fimbriate phenotype of a fimY mutant.This finding suggested that FimY acts upstream of FimZ to activate fimA expression (Zeiner et al. 2013).FimY and FimZ strongly activate each other's expression and weakly activate their own expression (Saini et al. 2009); however, the presence of both proteins is required IS118, MicM, as well as the sigma E-regulated sRNAs MicA (regulating fimB) and RybB (regulating fimA) (Bak et al. 2015;Gogol et al. 2011).
Knowledge of the environmental conditions that control the expression of a virulence lifestyle factor is important for relating and understanding the biological context that underlies an infection.Briefly, T1F is expressed by UPEC strains that colonize the bladder, but their expression is downregulated in bacteria that ascend to the kidneys (Snyder et al. 2005).Even though the pH, osmolarity and composition of urine depend on the nutritional state, hydration, and health, the urine composition can be altered during its passage through the urinary tract until it is stored in the bladder.This passage might create conditions suitable for T1F expression, in addition to the presence of mono-mannose rich uroplakin as a receptor in the bladder epithelium (Dalghi et al. 2020).Furthermore, the loss of aerobic respiration negatively impacts the expression.This phenomenon is observed in bacteria growing in biofilms, where the fim promoter of bacteria inhabiting deeper layers is phase-OFF, but it is switched ON in air-exposed bacteria on the surface of the biofilm, likely preparing them to be dispersed to other sites (Floyd et al. 2016).

Type 1 fimbria of Salmonella
T1F is also a device for bacteria that colonize the intestine, as the case of Salmonella enterica.T1F binds to mannosylated mucous components in the gastrointestinal tract facilitating colonization, and at the same time, it is considered a limiting factor in the spread of bacteria outside of the intestinal tract (Klasa et al. 2020;Kuzminska-Bajor et al. 2015).
Despite the morphological and functional similarities of T1F in E. coli and Salmonella, they are serologically and evolutionarily unrelated, resulting in different regulatory mechanisms of the fim operon in Salmonella compared to E. coli.The fim gene cluster of Salmonella comprises ten protein-encoding genes (fimA, fimI, fimC, fimD, fimH, fimF, fimZ, fimY, fimW, and stm0551 in the genome of S. Typhimurium), and one more encoding a tRNA-Arg (fimU) (Kolenda et al. 2019;Purcell et al. 1987).This T1F is a rodshaped structure mainly composed of FimA, followed by FimF, and finally FimH, the lectin-like protein positioned at the top of the pilus, which is associated with tissue tropism (Grzymajlo et al. 2017).Among the proteins encoded in the cluster, FimW, FimY, FimZ and the STM0551 open reading frame are notable for playing a prominent role in the transcriptional regulation of T1F, while tRNA-Arg additionally controls the expression of T1F at the translational level.
In E. coli, the underlying mechanism controlling the expression of T1F involves the inversion of the fimS switch, which is primarily mediated by the action of the FimB and 1 3 tRNA molecule that specifically recognizes the rare arginine codons, AGA and AGG.A high frequency of these codons is found within the regulatory fim genes, mainly in fimY, which contains five of these codons, three of them occurring before position 14.In the absence of fimU, the production of FimY is significantly affected; however, it could be restored to high levels when the gene is replaced or when the first three rare arginine codons are exchanged for major arginine codons.In this scenario, T1F production in S. Typhimurium is restored (Swenson et al. 1994;Tinker and Clegg 2001).Similar observations have been made for T1F of E. coli, where the disruption of the leuX tRNA leu (UUG) results in translational regulation of FimB, which affects the switch ON of T1F (Ritter et al. 1997).
Additionally, the product encoded by the stm0551 gene in S. Typhimurium, located between fimY and fimW, was found to downregulate the expression of T1F.STM0551 is an 11.4 kDa putative c-di-GMP phosphodiesterase (PDE) that exhibits PDE activity in vitro.In the absence of this protein, the bacteria exhibit constitutively active T1F production, and the expression of the fimA and fimZ genes increases in a mutant strain, even when grown on solid-agar medium, an unfavorable condition for the expression of T1F (Wang et al. 2012).Recently, it has been reported that the N-terminal portion of FimY contains amino acid residues that exhibit some similarity to those found in the proteins containing for high-level expression of fimZ (Yeh et al. 2002).Interestingly, pull-down assays revealed interactions between FimY and FimZ, leading to the hypothesis that FimY functions as a DNA-binding protein to activate fimZ and that the FimY-FimZ protein complex could regulate other fim genes (Wang et al. 2014).
Conversely to the roles of FimZ and FimY, FimW acts as a negative regulator of T1F, exhibiting a four to eightfold increase in fimbrial production in S. Typhimurium in the absence of the functional protein (Tinker et al. 2001).FimW represses transcription from the promoter of fimY within a negative feedback loop, since FimY activates the promoter of fimW (Saini et al. 2009).fimW expression in serovar Typhimurium increases under conditions that select for poorly fimbriated bacteria and low fimA expression (Tinker et al. 2001).A significant protein interaction between FimW and FimZ has been reported; however, this interaction occurs only when FimZ is phosphorylated, suggesting a possible interference with FimZ-mediated activation of fimA expression due to downmodulation of the active FimZ protein (Tinker et al. 2001;Zeiner et al. 2013).FimW also controls its own expression since the activity of the fimW promoter is increased in the absence of FimW protein (Tinker et al. 2001).
Regulation of the fim cluster also involves the modulation of FimY translation by the product encoded by fimU, a along with an increase in the amount of FimZ.This allows FimZ to bind more effectively to the fimA promoter, overcoming the weak binding of Lrp to motifs 2, 3, and 4 (Baek et al. 2011;McFarland et al. 2008).

Pap (P) fimbria
As described before, acute pyelonephritis occurs as a complication of an ascending UTI that spreads from the bladder to the kidneys and their collecting systems causing inflammation.The main causes of acute pyelonephritis are Gram-negative bacteria, with the most common being E. coli (Belyayeva et al. 2024).UPEC uses the pyelonephritisassociated pilus (Pap) to interact with glycosphingolipids on the surface of uroepithelial cells ascending to the kidneys and facilitating colonization (Legros et al. 2019).
The P pilus structure is complex and consists of a filament of 1-2 μm in length shaped by a thin tip fibrillum mounted on the pilus rod.The base of the pilus is composed of two subunits: PapA, the major subunit, and PapH, the termination rod subunit (Hospenthal et al. 2017).The pilus rod is connected with the tip fibrillum through PapK, an adaptor protein.Finally, the Pap tip is composed of the proteins PapE, PapF and the adhesin PapG which recognize Gal(α1-4)Gal moieties mainly found in kidney cells (Legros et al. 2019;Lund et al. 1987).The proteins PapC and PapD are involved in the assembly mechanism; they are the usher and the periplasmic chaperone, respectively (Lillington et al. 2015).The function of PapJ is unknown but it may be involved in protecting the pilus integrity during assembly (Tennent et al. 1990).
The P pilus is encoded by the gene cluster papIBAHCD-JKEFG; in addition to containing the structural components, it encodes for PapI and PapB, a small Lrp-binding protein and a local transcription factor, respectively (Hernday et al. 2002).papI is located divergently from the papBAHCD-JKEFG cluster.Due to this arrangement, the pap cluster is regulated by two promoters: PpapI, which activates the expression of papI and PpapBA, which controls the rest of the genes organized as an operon (Baga et al. 1985).The intergenic region between papI and papB comprises 330 bp and includes all the elements that are required for transcription to occur (Fig. 3).
The transcriptional activation of the pap cluster is subject to a complex and coordinated regulatory network that has been extensively studied and reviewed elsewhere (Blomfield 2001;Hernday et al. 2004b;Uhlin et al. 2000;Zamora et al. 2020).Here, we highlight the main aspects of the mechanism underlying pap regulation, which involves nucleotidebinding proteins H-NS and Lrp, the local transcriptional regulators PapI and PapB, and other elements such as CRP, RimJ, the TCS CpxRA and the sRNA PapR.Furthermore, the PilZ domain; one of the best-known c-di-GMP receptor domains.Although a change from arginine to alanine at position 7 decreases the expression of fimA and fimZ, secondary structure prediction analysis did not reveal similarity between FimY and PilZ-like proteins (Kuan and Yeh 2019).Further investigation is necessary to understand the role of c-di-GMP in the regulation of T1F in S. Typhimurium.
The TCSs PhoBR, QseBC, PhoPQ and BarA/SirA, where PhoR, QseC, PhoQ and BarA are the sensor histidine kinases, and PhoB, QseB, PhoP and SirA are the response regulators, have been implicated in the regulation of T1F.PhoBR, responsive to phosphate availability, is capable of inducing fimZ expression (Baxter and Jones 2015), while SirA binds directly to the fimA promotor region to control its expression (Teplitski et al. 2006).An intriguing, and yet unexplained, phenotype was observed for the qseB and qseC mutants of S. Typhi; in the absence of QseC, the transcription level of fimA is decreased, but no effect was seen in the absence of QseB (Ji et al. 2017).In addition, although PhoPQ does not directly affect fimZ expression, under low magnesium conditions, the PhoPQ regulon is activated, leading to the phosphorylation of FimZ (Baxter and Jones 2015).
T1F production is also regulated by transcription factors related to metabolism, the stress response or even the control of the expression of virulence lifestyle factors.A catabolite repression mechanism was described early on to regulate the T1F production in bacteria such as S. Typhimurium, several strains of E. coli, and Serratia marcescens (Kalivoda et al. 2008;Muller et al. 2009;Saier et al. 1978).This mechanism involves the participation of adenylate cyclases, cAMP, and CRP that respond to environmental carbon sources.In E. coli, where it has been more studied, CRP-cAMP plays a dual role, affecting both the phase variation process and fimA promoter activity, and even controlling the expression of Lrp (Muller et al. 2009); however, the exact mechanism controlling T1F in Salmonella remains unstudied.Moreover, the RNA polymerase-binding protein DksA has also been involved in the activation of fimA (Cohen et al. 2022).
As described before for T1F of E. coli, Lrp and H-NS are involved in transcriptional regulation of the fim cluster.Lrp exerts a dual role in modulating the expression of the fimA and fimZ promoters through direct binding, depending on the growth conditions and the amount of Lrp present.At low concentrations, Lrp acts as a transcriptional activator, whereas the overproduction of Lrp abrogates T1F expression.There are four binding sites for Lrp upstream of fimA where it binds independently of FimZ.It has been proposed that the binding of Lrp to sites 2 and 3 aids the binding of H-NS, excluding the binding of FimZ and keeping the expression of fimA repressed.On the other hand, for transcription to occur, the binding of Lrp to site 1 is necessary, 1 3 Under this condition, Lrp acts as a transcriptional repressor independently of H-NS (van der Woude et al. 1995).
Conversely, Lrp can also activate the transcription of pap when it binds to distal sites; however, this happens in conjunction with PapI (Hernday et al. 2003).Once H-NS is not repressing, the activity of the two promoters depends on the concentration of the local regulators PapI and PapB.At a basal concentration of PapI, Lrp binds with the highest affinity to the proximal sites (1, 2 and 3) on the papBA promoter, where it acts as a repressor.However, with an increase in PapI, the affinity of Lrp for sites 1-3 decreases, and its affinity for the distal sites 4-6 increases, facilitating the transcription of papBA and its transition it to an active state (Nou et al. 1995).
DNA methylation plays both positive and negative roles in the transcription of pap.In the papBA regulatory region there are two GATC sites located in the middle of Lrp sites 2 and 5, respectively, which are recognized by Dam methylase.Methylation of the GATC distal site occurs in the inactive state (phase OFF) when Lrp is bound to proximal sites under conditions of low PapI concentrations, whereas methylation of the proximal GATC site occurs in the active the expression of these structures is inherited epigenetically, controlled by a methylation mechanism dependent on deoxyadenosine methylase (Dam) (van der Woude et al. 1996).As a result, P fimbriae are subject to a phase variation control mechanism, in which cells are either piliated (phase ON) or non-piliated (phase OFF) (Blyn et al. 1989).
As with other fimbriae, the expression of P fimbriae is controlled by environmental cues (Blyn et al. 1989;Zamora et al. 2020).H-NS binds to the entire intergenic region, forming a repressive nucleoprotein complex under conditions such as low temperature, high osmolarity, the presence of glucose as a carbon source and rich medium (White-Ziegler et al. 2000).Therefore, the activation of pap requires the displacement of H-NS, which is achieved through the action of CRP in cooperation with Lrp (Forsman et al. 1992).
Lrp plays a dual role in the transcriptional regulation of pap in a leucine-independent manner (Braaten et al. 1992).This protein can bind to six sites on the papAB promoter: the proximal sites 1, 2 and 3 and the distal sites 4, 5 and 6.Lrp binding at site 3 covers the − 35 and − 10 sequences, preventing the interaction with the RNA polymerase and maintaining the transcription of papBA in an inactive state.increases UPEC adhesion to bladder and kidney cell lines independently of T1F (Khandige et al. 2015).
An interesting regulatory crosstalk among the different C-U gene clusters has been observed, primarily controlling unnecessary expression of surface structures at times (Holden et al. 2006;Lopez-Garrido and Casadesus 2012;Sjostrom et al. 2009).These regulatory mechanisms are essential during infection for controlling tropism to specific colonization sites, evading the host immune system and avoiding unnecessary energy use.Furthermore, expression of fimbriae is often inversely correlated with flagellar expression (Lane et al. 2007;Simms and Mobley 2008), allowing bacteria to either establish contact with a specific site (sessile lifestyle) or move to a new site (planktonic lifestyle).
An intriguing communication between P pilus and T1F is primarily mediated by PapB.PapB's role in fim transcription is predominantly negative; it binds to fimS, inhibiting phase transition by FimB in both directions by locking the fim switch, and enhancing FimE activity by improving fimE expression (Holden et al. 2006;Totsika et al. 2008;Xia et al. 2000).While the mechanism of crosstalk has been associated with other members of the PapB family, it is mainly restricted to the P and S fimbriae with T1F (Holden et al. 2001).As mentioned previously, bacterial tropism is facilitated by the expression of at least two different fimbriae in UPEC: T1F and P pilus.T1F recognizes mannosylated proteins in the bladder, while P pilus bind to Gal(α1-4)Gal moieties in the kidney.Thus, coordinated expression ensures the sequential expression of adhesins during urinary tract infections.

Concluding remarks
Nearly four decades of research have laid the molecular groundwork for understanding the mechanisms controlling the expression of the fim and pap fimbrial operons, which encode prototypical members of the C-U family.These operons have been invaluable models for describing various aspects from fimbriae structure and biogenesis to their critical role in host-pathogen interactions, including adhesion and cellular tropism, especially in urinary tract infections caused by UPEC.
The genomic era and the study of clinical isolates have further enriched our understanding by revealing how genetic variations diversify functional roles and regulatory mechanisms.However, much remains to be learned about how these mechanisms impact the success of an infection and how individual host conditions influence infection outcomes.Recent in vitro studies, for example, show that planktonic bacteria growing in human urine express little state (phase ON) when the PapI concentration is higher and Lrp is bound to the distal sites (Braaten et al. 1992;Hernday et al. 2003).Interestingly, Lrp binding at the proximal or distal sites is mutually exclusive (Hale et al. 1998), and methylation of the proximal GATC site is required for phase ON.Dam methylation at the proximal GATC site has a positive effect because it blocks the PapI-dependent increase in the affinity of Lrp for sites 1-3, thereby favoring binding to distal sites and enabling the transcription of pap (Hernday et al. 2003).
The second locally encoded transcription factor is PapB, which belongs to the family of adhesin regulators with DNA-binding activity.It primarily plays a positive role in pap transcription; however, at high concentrations, PapB has been reported to exhibit repressor activity in bacteria (Hernday et al. 2002;Holden et al. 2001).Three binding sites for PapB have been identified: site 1 spans from 40 to 90 bp upstream of the papI promoter, site 2 overlaps with the papBA start site, and the third site is located within the papB coding sequence.PapB exhibits a higher affinity for site 1, where it oligomerizes between the papI start site and a CRP binding site (Forsman et al. 1989).It has been proposed that CRP interacts with PapB, and that both stabilize the binding of the RNA polymerase to the papBA promoter, allowing its own transcription (Weyand et al. 2001).Conversely, the binding of PapB within sites 2 and 3 likely represses papBA expression by steric hindrance of RNAP (Xia and Uhlin 1999).
The transcriptional regulation of the pap fimbrial operon is also influenced by various systems, including the TCS CpxRA.When the synthesis of P fimbriae is dysregulated, the fimbria subunits misfold and form aggregates in the periplasm.Under this stress condition, it was found that the response regulator CpxR has a positive modulatory effect on pap phase variation by maintaining the phase-ON state and that phosphorylated CpxR specifically binds to the pap promoter (Hung et al. 2001).However, in a contrasting observation, it was later reported that CpxR, by competing with Lrp for binding, blocks the Pap phase OFF to ON switch and pap transcription (Hernday et al. 2004b).An additional regulator of pap is RimJ, an N-Acetyltransferase of ribosomal protein S5, downregulates the transition into the phase ON state in response to various environmental stimuli such as low temperature, rich medium conditions, and glucose presence.The specific mechanism by which RimJ regulates these processes remains unknown, but it has been proposed that CRP and Lrp could serve as substrates for its N-acetyltransferase activity (White-Ziegler et al. 2002).Moreover, an Lrp-regulated virulence-associated trans-acting small RNA called PapR was found to mediate the posttranscriptional repression of papI.Deletion of PapR 1 3 to no T1F, while bacteria associated with bladder epithelial cells are highly fimbriated (Greene et al. 2015;Staerk et al. 2016).This potentially explains why clinical isolates of UPEC did not express T1F in the urine of women with cystitis, even though its expression significantly increased in a mouse model of urinary tract infection (Hagan et al. 2010).Moreover, these observations are underscored by studies in other animal models, such as pigs, which revealed that the infectious potential of UPEC relies on T1F to overcome initial infection bottlenecks and generate an early immune response, only to become dispensable in later infection stages where regulatory crosstalk may play a crucial role in infection progression (Staerk et al. 2021).
Further exploration of fimbrial operon regulation in in vivo physiological settings is essential to deepen our understanding of these regulatory networks.This knowledge could pave the way for targeted interventions and improved clinical outcomes in the prevention and treatment of UTIs.

Fig. 1
Fig. 1 Schematic representation of the regulatory network controlling the expression of the fim operon in E. coli.The transcription of the fimAICDFGH operon is regulated by the phase-ON (top right-bound arrow) and phase-OFF (bottom left-bound arrow) orientations of the fimS switch, which contains the fimA promoter.fimS is flanked by the inverted repeat left (IRL) and right (IRR) sequences, which are depicted as gray ovals.Upstream of the fimS region are the genes encoding the FimB and FimE recombinases, which bind to the IRL and IRR to control phase variation of T1F by fimS DNA inversion.Additional recombinases encoded outside the fim cluster are also illustrated.Several transcriptional regulatory factors positively (green arrows) or

Fig. 2
Fig. 2 Schematic representation of the regulatory network controlling the expression of the fim operon in Salmonella.The transcription of the fimAICDHF operon is regulated by multiple factors that act in concert.Downstream of the fim operon are the genes encoding FimZ, FimY, FimW and the PDE STM0551, depicted as light blue, indigo, dark blue and orange rounded squares, respectively.H-NS is represented by yellow ovals, whereas Lrp binding sites are denoted by purple circles.Several transcriptional regulatory factors positively (green arrows)